MERHAB 2010: Project Summaries

Institution: University of South Carolina and University of Delaware

Investigators: D. Greenfield and K. Coyne

Abstract

Introduction to the Problem: A shift from light microscopy to molecular approaches for quantifying harmful algal bloom (HAB) species has been driven by the need to expedite sample processing while increasing detection sensitivity and accuracy.  Cell homogenate approaches have been developed to quantify HAB species using quantitative real-time PCR (QPCR) and sandwich hybridization (SHA). With QPCR, species are enumerated by enzymatic amplification of DNA.  SHA directly detects RNA from and unpurified/unamplified homogenate. Both methods have been validated for HAB species quantification.  However, they have not been thoroughly compared, representation a key gap in the ability to provide recommendations to managers for a specific regulatory requirement.  The project provides a thorough assessment of QPCR and SHA for HAB monitoring and research using laboratory and field studies.
Rationale and Management Relevance: Multiple initiatives have contributed to the development and validation of HAB detection and quantification technologies in laboratory and in situ formats. Despite these advances, fundamental questions remain surprisingly unanswered: (1) Will each technique provide comparative quantitative results? (2) Do cell growth and nutrient conditions affect data interpretation? (3) Does calibration using laboratory cultures translate to estimates of natural field populations? (4) From a resource manager standpoint, which quantification technique is most suitable for a particular monitoring need and budget?  Addressing the need for cross-method comparisons was identified as a critical priority by the HAB community to provide decision makers with information necessary to enhance regional monitoring.  Work herein directly addresses these priorities by engaging managers, local communities, and researchers to provide a targeted assessment of two molecular based quantification methods (QPCR and SHA) for HAB research and development.
Scientific Objectives: Evaluations used herein are applicable to several HAB species, but the proposed study will focus on the globally-distributed harmful raphidophyte, Heterosigma akashiwo as a model species. Objectives include (1) Directly compare QPCR and SHA for quantification of H. akashiwo isolates spanning a range of cell abundances, growth phases, and nutrient conditions; (2) determining the extent to which quantification of H. akashiwo is comparable using QPCR and SHA for natural phytoplankton communities (3) Synthesize comparisons according to a suite of criteria to enhance HAB monitoring and research activities.
Approach: In this targeted study, QPCR and SHA will be critically compared , using microscopy as a “gold standard,” to provide recommendations to managers for HAB monitoring strategies based upon multiple criteria: (1) range and limit of detection, (2) accuracy and specificity, (3) cost/sample, (4) initial investment and equipment maintenance cost, (5) sample throughput, (6) applicability to live and preserved samples, (7) circumstances when one method would be preferable over another. Comparisons will focus on H. akashiwo representing a range of geographic regions, cell growth phases, and nutritional conditions in laboratory and field studies.
Expected Outputs/Outcomes: Resource managers will be provided with necessary tools to make informed decisions about appropriate method(s) for individual HAB monitoring needs and budgets.  Information will be conveyed through various outputs: workshops, a website, publications, and scientific presentations.  Outcomes will include: improved knowledge for management decisions, and changes in management behavior as method(s) are incorporated into HAB Monitoring programs.